Ion exchange cryptands covalently bound to substrates

ABSTRACT

One embodiment of the invention comprises an ion exchange composition formed by reacting unsaturated carbon to carbon moieties pendant from derivatized ion binding cryptands with a support substrate under free radical activation conditions to form a covalent bond therebetween. 
     In another embodiment, a cryptand ion exchange composition is made by covalently bonding unsaturated carbon to carbon moieties pendant from a derivatized ion binding cryptands with unsaturated carbon to carbon moieties pendant from a support substrate under free radical activation conditions to form covalent bond.

FIELD OF THE INVENTION

The present invention relates to cryptands covalently bound to a supportsubstrate for uses such as in ion exchange chromatography.

BACKGROUND OF THE INVENTION

Cryptands and other macrocyclic compounds such as crown ethers,spherands, cryptahemispherands, cavitands, calixarenes, resorcinorenes,cyclodextrines, porphyrines and others are well known. (ComprehensiveSupramolecular Chemistry Vol. 1-10, Jean-Marie Lehn—Chairman of theEditorial Board, 1996 Elsevier Science Ltd.) Many of them are capable offorming stable complexes with ionic organic and inorganic molecules.Those properties make macrocyclic compounds candidates for variousfields, for instance, catalysis, separations, sensors development andothers. Cryptands (bicyclic macrocycles) have extremely high affinity tometal ions. The cryptand metal ion complexes are more stable than thoseformed by monocyclic ligands such as crown ethers (Izatt, R. M., et al.,Chemical Reviews 91:1721-2085 (1991)). This high affinity of thecryptands to alkaline and alkaline earth metal ions in water makes themsuperior complexing agents for the processes where strong, fast andreversible metal ion binding is required. Examples of these processesinclude separation, preconcentration and detection of metal ions,analysis of radioactive isotopes, ion-exchange chromatography,phase-transfer catalysis, activation of anionic species and others.

Adding moieties with functionality to macrocyclic compounds permitsbinding of the derivatized macrocycles onto support substrates toprovide surface functionalization. Physical adsorption and covalentattachment are two common methods of binding. Cryptand adsorbed polymershave been reported as stationary phases for ion exchange chromatography(Lamb, J. D., et al., J. Chromatogr., 482:367-380 (1989); Niederhauser,T. L., et al., Journal of Chromatography A, 804:69-77 (1998); Lamb, J.D., et al., Talanta, 39(8):923-930 (1992); and Smith, R. G., et al.,Journal of Chromatography A, 671:89-94 (1994).

The majority of adsorbed materials have limited number of applicationsdue to their incompatibility with the solvents that elute the adsorbedfunctional layer. There is also a restriction on using these materialsat elevated temperatures. Covalent attachment reduces these problems.Previously reported substrates with covalently attached macrocyclesinclude silica gel, polymeric resins, thin films and others (Blasius,E., et al., Pure & App. Chem. 54(11):2115-2128 (1982); Montanari, F., etal., British Polymer Journal, 16:212-218 (1984); U.S. Pat. No. 5,393,892to Krakowiak, et al.; U.S. Pat. No. 4,943,375 to Bradshaw, et al.; U.S.Pat. No. 5,968,363 to Riviello, et al.; JP Patent No. 55018434A2 toKakiuchi, et al.; JP Patent No. 59145022A2 to Fujine, et al.; JP PatentNo. 61033220A2 to Fujine, et al.; JP Patent No. 4346064A2 to Watanabe,et al.; and PCT Publication W099/28355 to Darling, et al.

Many strategies for the synthesis of macrocyclic compounds have beendeveloped over the years (Comprehensive Supramolecular Chemistry Vol.1-10, Jean-Marie Lehn—Chairman of the Editorial Board, 1996 ElsevierScience Ltd.; Krakowiak, K. E., et al., Israel Journal of Chemistry32:3-13 (1992); Bradshaw, J S., et al., “Aza-Crown Macrocycles,” TheChemistry of Heterocyclic Compounds, Vol. 51, ed. Taylor, E. C., Wiley,New York, 1993; Haoyun, A., et al., Chemical Reviews 92:543-572 (1992)).However, the synthesis of functionalized macrocycles is difficult.Hydroxy, amino and carboxylic groups added to linear precursors beforethe ring closure step are commonly used functionalities forderivatization of macrocycles. Most of the synthetic procedures implyprotection of these groups prior to cyclization. Protected groups arechemically transformed into desired functions after the macroring isconstructed (Krespan, C. G., Journal of Organic Chemistry 45:1177-1180(1980); Montanari, F., et al., Journal of Organic Chemistry 47:1298-1302(1982); Haoyun, A., et al., Journal of Organic Chemistry 57:4998-5005(1992)). This methodology can impose considerable limitations onsynthesis and purification of functionalized macrocycles, especiallybicyclic and polycyclic compounds. Synthetic difficulties can lead tolow overall yields and high production costs of these materials.

Macrocyclic compounds containing allylic functionalities are known fromprior art (Krakowiak, K. E., et al., Journal of Heterocyclic Chemistry27:1011-1014 (1990)). Some of them were further hydrosilated andattached to silica solid supports (Bradshaw, J. S., et al., Pure & Appl.Chem. 61:1619-1624 (1989); Bradshaw, J. S., et al., Journal of InclusionPhenomena and Molecular Recognition in Chemistry 7:127-136 (1989)). Thesynthesis of allyl containing [2.2.2] cryptand 1 has been reported(Babb, D. A., et al., Journal of Heterocyclic Chemistry 23:609-613(1986)).

The methods for covalent attachment of the cryptands to polymericsubstrates are based mostly on the interaction of active layer of asubstrate, for example, benzyl chloride groups with hydroxyl or aminofunctionalized cryptand molecules (Montanari, F., et al., J. Org. Chem.,47:1298-1302 (1982); Montanari, F., et al., British Polymer Journal,16:212-218 (1984) and Montanari, F., et al., Tetrahedron Letters, No 52,5055-5058 (1979)). This interaction also involves the sideprocess—formation of the quaternary centers from the tertiary nitrogensof the macrocycle (Montanari, F., et al., British Polymer Journal,16:212-218 (1984). Quaternisation causes extended decomposition of themacrocycle via Hofmann degradation reducing the capacity of the anionexchange stationary phase. An amide group is another linker reported fora covalent functionalization of the substrates with cryptand molecules(Montanari, F., et al., British Polymer Journal, 16:212-218 (1984).Amides do not withstand the extremely high pHs used in anion exchangechromatography. Moreover, most of the described synthetic for producinghydroxyl or amino functionalized cryptands, are not practical to satisfythe requirements of industrial scale production.

There is a need to provide an improved method for covalent bonding ofcryptands to a substrate for uses such as a chromatographic separationmedium to separate anions.

SUMMARY OF THE INVENTION

One embodiment of the invention comprises an ion exchange compositionformed by reacting unsaturated carbon to carbon moieties pendant fromderivatized ion binding cryptands with a support substrate under freeradical activation conditions to form a covalent bond therebetween.

In another embodiment, a cryptand ion exchange composition is made bycovalently bonding unsaturated carbon to carbon moieties pendant from aderivatized ion binding cryptands with unsaturated carbon to carbonmoieties pendant from a support substrate under free radical activationconditions to form covalent bond.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an ion exchange compositionaccording to the present invention.

FIGS. 2 and 3 are chromatograms illustrating uses of the ion exchangecomposition of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect of the present invention, an ion exchange composition isformed by reacting unsaturated carbon to carbon moieties pendant from aderivatized ion binding cryptand C with a support substrate under freeradical activation conditions.

In one embodiment, the cryptand pendant unsaturated carbon to carbonmoiety is on a styrenic group which has been appended to the cryptand bytechniques such as described below. The properties of cryptands are wellknown as ion exchange compositions. As set forth in the publicationsdescribed above, cryptands bind with cations such as potassium, sodium,rubidium, calcium, strontium, barium, thallium and to a lesser degreewith cesium, magnesium and lithium and form an anion exchanger accordingto well known principles. The concentration of complexed cation isdirectly proportional to the binding constant K, also known as thestability constant. If these macrocycles are attached to a support suchas a conventional chromatographic stationary phase, and subsequentlycomplexed with certain cations, anion exchange chromatography can beachieved.

One advantage of the reaction schemes of the present invention is thatindividual multiple (e.g., 4×10¹⁴ to 4×10¹⁶ or more) strands ofderivatized macrocycle compounds (e.g., cryptands) per square meter canbe bound to each support particle and projecting therefrom asillustrated in FIG. 1. This provides a substantial quantity ofmacrocycle compounds in a format which is readily accessible to theapproach of both cationic and anionic species in an aqueous environment.Prior art based on adsorbed macrocycle compounds provides very limitedcapacity due to the restriction that this species must be adsorbed as amonolayer on a hydrophobic surface. Presenting the macrocycle compoundsas a brush polymer extending into the aqueous solution surrounding eachsupport particle, permits significantly better diffusion kinetics thancan be observed with adsorbed monolayer coatings of macrocyclecompounds, while at the same time allowing for significantly highercapacity than is possible with an adsorbed monolayer coating.Furthermore, positioning the macrocycle compounds in the proximity of ahydrophobic surface compromises the chromatographic performance ofmaterials based on adsorbed macrocycle compounds. Anionic hydrophobiccompounds exhibit poor chromatographic efficiency when that retentionsite is located in the proximity of a hydrophobic surface unless organicsolvent is added to the mobile phase. Of course, incorporating anorganic solvent into mobile phase flowing past the support particles isincompatible with the prior art adsorbed monolayer coatings because suchorganic solvent will slowly wash the adsorbed coating away. While thecomposite material derived from support particles which had beencovalently derivatized with macrocycle brush polymers are compatiblewith organic solvents, the high cost of organic solvents and the highcost of disposal of organic solvents make the use of organic solventshighly undesirable. Thus, the brush polymer configuration describedabove provides superior chromatographic properties without the need forthe addition of organic solvent and the incumbent disadvantages of itsuse.

In one preferred embodiment, the covalent bond is formed between thecryptand pendant moieties and an unsaturated carbon to carbon moietypendant from the polymeric substrate under free radical conditions. Forexample, the cryptands can be bound to resin beads such as cross-linkedpolystyrene resin for use in a packed bed for an ion exchange column.The form of the bed and the characteristic composition of the resin canbe of the type sold by Dionex under the IONPAC® product line. In aparticularly effective ion exchange composition of this type, thecryptand is derivatized to include a pendant styrenic group which formsthe covalent bond.

In a further embodiment, the only ion binding moiety bound to thepolymeric substrate is a cryptand.

The vinyl moiety of the styrenic terminal group is readily attachedunder free radical conditions to polymeric substrate particles includingterminal groups with ethylenic unsaturation as described in U.S. Pat.No. 5,865,994.

In another embodiment, the support substrate includes hydrogen atomswhich are abstractable under hydrogen abstraction conditions to form acovalent bond with the cryptand pendant unsaturated moiety. Suitableconditions for this reaction include free radical activation asdisclosed in U.S. Pat. Nos. 6,074,541 and 5,792,331.

An anion exchange column of the foregoing type can perform a variety ofanion exchange separations ranging from common anions such as fluoride,chloride, sulfate, nitrite, bromide, phosphate and nitrate; hydrophobicanions such as thiosulfate, iodide, thiocyanate and perchlorate; organicacids such as formic, acetic, glycolic, quinic acids. Polyvalent ionssuch as polyphosphates, oligonucleotides, and oligosacarrides at high pHcan also be separated with this column.

The conditions for separation of anions using cryptands are well known.(Lamb, J. D., et al., J. Chromatogr., 482:367-380 (1989); Niederhauser,T. L., et al., Journal of Chromatography A, 804:69-77 (1998); Lamb, J.D., et al., Talanta, 39(8):923-930 (1992); and Smith, R. G., et al.,Journal of Chromatography A, 671:89-94 (1994). An example is shown inFIG. 2 where the separation of seven common anions is accomplished usingconventional suppressed anion exchange techniques. Here we see theseparation of fluoride (peak 1), acetate/formate (peak 2), chloride(peak 3), sulfate (peak 4), nitrite (peak 5), bromide (peak 6),phosphate (peak 7), and nitrate (peak 8) using a mobile phase of 70millimolar sodium hydroxide.

As set forth above, in one embodiment a monomeric cryptand is graftedonto the support substrate (stationary phase) using a free radicalinitiator to produce a covalently bound functionality. This stationaryphase is stable when used with aqueous or organic mobile phases.Bicyclic and tricyclic aliphatic and aromatic cryptand moleculescontaining nitrogen and oxygen heteroatoms or only oxygen heteroatoms oronly nitrogen heteroatoms or incorporated heterocyclic fragments formetal ion binding can also be applied as modifiers. Other polymerizablecryptand compounds such as their styrenic, allylic, acrylic, methacrylicand any other alkene and alkyne derivatives can be used for grafting.Once the cryptand is attached by a covalent bond by this or any of themethods possible to form such bonds, the cryptand can now be utilized asa stable functional group for anion exchange chromatography.

Anion exchange mobile phases are typically basic solutions such assodium or potassium hydroxide (or carbonate). Elution of anions occursvia competition between the mobile phase anion such as hydroxide orcarbonate and the sample anions for cation exchange sites on thestationary phase. In conventional suppressed anion exchangechromatography, the associated cation typically plays a negligible rolein the process. In cryptand based anion exchange, the cation plays acrucial role. It generates the anion exchange site, and depending on thecation, determines the capacity and selectivity of the stationary phase.

For example, if a basic mobile phase such as potassium hydroxide is usedwith [2.2.2] cryptand resin, a high capacity stationary phase is createdsince potassium has a high binding constant relative to sodium orlithium. If a mobile phase such as sodium hydroxide is used, a lowercapacity stationary phase is now created since sodium has a lowerbinding constant. A low capacity phase is generated if lithium hydroxideis used as the mobile phase since lithium has a low affinity for the[2.2.2] cryptand. The capacities decrease as expected based on thebinding constants of the 2,2,2 cryptand with potassium, sodium, andlithium cations.

According to the present invention, the macrocycle is permanentlyattached to a support substrate and will not slowly bleed off an ionexchange column when using aqueous eluents or eluents which containsolvents.

Another advantage is the ability to rapidly restore columns whoseperformance has been compromised by contamination with polyvalent ions.This can be done by using a very low capacity mode with no substantialaffinity for these ions. Under these conditions, the polyvalentcontaminants easily elute off the column and the original performance isrestored.

Conventional anion exchange columns have a fixed capacity andselectivity, which cannot be adjusted. In the present invention, one hasthe ability to vary the anion exchange capacity by changing the cationcomplexed with the cryptand. Selectivity to a lesser degree can also beadjusted by these mobile phase changes. This feature allows the user tomodify the column performance relative to the sample being analyzedsimply by the choice of mobile phase. Not only can the capacity beadjusted for a single analysis, but it can also be varied during ananalysis by using a higher capacity mobile phase at the beginning andthen changing to a lower capacity format by either a step change or agradient. An example of this is shown in FIG. 3 where the initial mobilephase is sodium hydroxide and then a step change is made to a lithiumhydroxide mobile phase, which is a very low capacity form. The firstpeaks eluting off are low valency polyphosphates and the run ends withthe elution of high valency ions.

The benefit of this approach is that it allows one to elute very highvalency ions without having to increase the mobile phase ionicconcentration. The total ionic concentration throughout the run is only30 mM, a relatively low concentration. This technique is known as“gradient capacity ion chromatography” (2,3) chromatography (Lamb, J.D., et al., J. Chromatogr., 482:367-380 (1989); Lamb, J. D., et al.,Talanta, 39(8):923-930 (1992);

Traditional anion exchange columns require very high concentrationmobile phases when analyzing polyvalent ions such as polyphosphates,oligosacarrides, oligonucleotides, etc. The concentrations neededsometimes exceed the ability of a suppressor to suppress. By using acryptand based column one can eliminate large baseline changes oftenseen with steep gradients or step changes with conventional approaches.

One mode for forming a derivatized cryptand is disclosed in Bordunov, etal. application, entitled “A Derivatized Macrocycle Compound forCovalent Bonding to a Substrate and Method of Forming and Use,” filedsimultaneously herewith. As disclosed therein, cryptands C arederivatized by the use of a sulfur-containing derivatizing agent to forma product which includes a terminal functional moiety R bound, directlyor indirectly, to C. R is capable of covalently binding to a supportsubstrate or of being converted into a form capable of covalentlybinding to such a substrate. In general, the derivatized C has thefollowing structure:

-   -   wherein C is a molecular framework monocyclic or polycyclic        cryptand moiety containing at least 12 atoms in each cycle;    -   L is a substituted or unsubstituted carbon chain linker        covalently bound to C including at least one carbon atom in a        structure selected from the group consisting of an aliphatic,        aromatic or heterocyclic linker including heteroatoms        substituted for hydrogen atoms on the linker;    -   R is a terminal functional moiety capable of covalent binding to        a support substrate or of being converted into a functional        moiety capable of covalent binding to a solid support substrate;    -   X and Y are moieties selected from the group consisting of        protons, aliphatic groups, aromatic groups, optionally including        heteroatoms, substituted for hydrogen atoms in the moieties,        selected from the group consisting of oxygen, nitrogen, sulfur,        or phosphorus heteroatoms; and S is sulfur.

Suitable macrocyclic compounds C are monocyclic, bicyclic, tricyclic orpolycyclic molecular frameworks. Examples of such macrocyclic compoundsinclude crown ethers, cryptands, spherands, cryptahemispherands,cavitands, calixerenes, resorcinorenes, cyclodextrines and porphyrinessuch as of the type described above. According to the invention, the Rgroup in structure (1) can be covalently bound in one or more steps to asupport substrate Z to form the following structure:

the transformation of R or functional group derived from R results inthe formation of linker R₁ during functionalization of Z. R₁ is acovalent linker between S and Z. It can be a substituted orunsubstituted carbon chain including at least one carbon atom in astructure selected from the group consisting of an aliphatic, aromaticor heterocyclic linker including heteroatoms substituted for hydrogenatoms on the linker. Although R₁ is illustrated to be directly bound toZ, it can be bound to an intermediate compound which is capable ofcovalent binding to Z, as shown in reactant scheme (5). Thus, the -R₁-Zlinkage of structure (3) encompasses a direct and indirect bonding anddoes not exclude such an intermediate linkage.

Any support substrate Z can be used so long as C in structures (1)-(3)is capable of performing its desired function, e.g., to serve as an ionexchanger. One form of structure (3) is a packed bed of particles ofderivatized macrocycle compound covalently bound to substrate Z.Suitable substrates include organic or inorganic materials such ascross-linked and uncross-linked polymers, resins, organic or inorganicmonoliths, sol-gels, other forms of gels such as silica gels, inorganicsupports such as zeolites, aluminum oxide, titanium dioxide, zirconiumbased supports, glasses, carbon black, activate carbon, carbonnanotubes, fibers, pyrolized materials, organic and inorganic crystals,liquid crystals, colloids, nanoparticles, organic and inorganic gels,latexes, foams, membranes and films. Also, Z may be in the form ofmonolayers such as surfaces of chips, silicon wafers, the walls ofcapillaries used for gas, liquid, capillary and ion exchangechromatography, capillary electrophoresis, separation, extraction, solidphase extraction, filtration, purification, transport, complexation,molecular and ion recognition, concentration, sensing an analysis oforganic and inorganic molecules and ions and also for catalysis, phasetransfer catalysis, solid phase synthesis or for other applications.

One particularly useful macrocycle comprises a cryptand bound to asupport substrate such as resin copolymer particles in a flow-throughion exchange bed, e.g., using the cryptand functional bed for anionexchange chromatography.

According to one embodiment of the present invention, a macrocycliccompound C is derivatized to include a pendant reactive moiety such asan allylic group by well known methods as described above. C is definedto include such reactive moieties which are capable of bonding to HSR asdescribed below. Thus, the HSR reagent is covalently bound to C to forman intermediate product of the type shown in structure (1) in which theR group is covalently bound to the macrocycle indirectly through thesulfur atom S. As set forth above, the R group can be in a functionalform suitable for direct or indirect covalent attachment to the supportsubstrate in a single or multiple steps. Scheme (4) illustrates thederivatization of macrocyclic compound (2) with HSR under the conditionsof free radical addition. In a particular case compound (2) is thecryptand with the allylic pendant moiety.

In structure (1), S is connected to the macrocycle C through theintermediate linker L including at least one carbon atom. In structure(2), L is disposed between C and an unsaturated carbon to carbon bond Uwhich interconnects L and the terminal carbon atom bound to Y. Thepurpose of linker L is to incorporate function U into the macrocycle C.

In one embodiment, the unsaturated carbon to carbon bond, e.g., a —C═C(double bond) or —C/C— (triple bond) described as U in (2) serves as thereacting site for free radical addition of HSR to C through the terminalgroup U distal to C. The unsaturation is preferably provided by thedouble bond, e.g., a terminal allyl group. Linker L may be attached to Cat any site that does not significantly affect the ability of themacrocycle to provide the desired function, e.g., to complex with an ionof interest. Thus, for a cryptand, the attachment would notsignificantly affect binding of the cation or its associated anion. Asdescribed above, the backbone of the linker L is preferably from about 1to about 20 atoms in length, preferably from 3 to 8 atoms in length. Thelinker chain may be straight chained or branched and it may also includesaturated or unsaturated carbon atoms for heteroatoms substituted forhydrogen atoms on the linker including oxygen, nitrogen, sulfur orphosphorus. Usually the linker group will contain from 1 to 3heteroatoms. The heteroatoms may be placed in the linker chain atpositions where they will have no significant adverse affect on the ionseparation characteristics of the composition. The linker group L can besimilar to the corresponding linker L in U.S. Pat. No. 5,865,994,incorporated herein by reference.

Conditions suitable for free radical attachment of the HSR group to apendant unsaturated group on the macrocycle by free radical initiationare well known in the art. For example see Griesbaum, K, Angew. Chem.Internat. Edit. Vol.9, No.4, 273-287 (1970).

In one embodiment of the reaction scheme (4), R is in a form capable ofdirect covalent attachment to a support substrate without converting Rto a form capable of covalent attachment. The conversion of R such asprotection/deprotection reactions might be necessary to keep R intactduring the reaction (4). Another reason for an optionalprotection/deprotection of R is to prevent the interference of group Rwith the course of reaction (4). The example of theprotection/deprotection of R is using a carboxylic acid in a form ofester protected R group in reaction (4) followed by its conversion(deprotection) to carboxylic acid upon hydrolysis, prior to itsattachment to a substrate. The groups R suitable for a direct covalentattachments to a support substrate with the possible use ofprotection/deprotection include proton, amines, epoxides, aldehydes,ketones, alcohols, phenols, thiols, carboxylic acids, thiocarboxylicacids, amides and esters of carboxylic and thiocarboylic acids,phosphoric and phosphoric acids, esters of sulfonic acids.

Reaction schemes such as reported earlier (Montanari, F., et al., J. OrgChem., 47:1298-1302 (1982); Montanari, F., et al., British PolymerJournal, 16:212-218 (1984) and Montanari, F., et al., TetrahedronLetters, No 52, 5055-5058 (1979)) may be used for a directfunctionalization of a support substrate with the cryptand modifier. Oneof the described approaches is the reacting hydroxymethyl functionalizedcryptand with the chrolomethyl polystyrene polymer in presence of thebase. Some disadvantages of this and other previously reported methodsfor a direct covalent attachment of the cryptands are described above.

There are at least two ways of indirect attachment of (1) to a supportsubstrate. The first method is the conversion of R to a group capable ofcovalent binding to a substrate under non radical conditions to formstructure (3). The example of this approach can be a conversion of (1)where R is the alcohol moiety. The alcohol group can be easilytransformed into a tosyl or mesyl derivative which reacts with thedeprotonated hydroxyl groups of a support substrate to form a covalentlink R₁. Functional groups that can be prepared by conversion of R forthe further non radical covalent attachment to a substrate includeamines, epoxides, aldehydes, ketones, alcohols, phenols, thiols,carboxylic acids, thiocarboxylic acids, amides and esters of carboxylicand thiocarboxylic acids, phosphoric and phosphoric acids, esters ofsulfonic acids, acyl halides, alkyl and aryl halides and activatedcarboxylic acids.

A second method of indirect attachment of (1) to a support substrate isthe conversion of group R into polymerizable moiety followed by itscovalent binding to a substrate under free radical conditions. Theindirect attachment of the below specific reaction scheme (5)illustrates a two-step procedure in which —SR in the HSR reagent firstis bonded through linker L to C. Then, in a second step, the bound R isreacted with another reagent to form a pendant group on R capable ofcovalent binding to a substrate via radical process. In the followingspecific reaction scheme (5), the pendant group is an ethylenicallyunsaturated (vinyl) group which can be bound to the support substrateunder free radical activation conditions.

Referring specifically to reaction scheme (5), an allyl derivative of[2.2.2] cryptand 1 is first formed by known chemistry as describedabove. Then, it is covalently bound to the HSR reagent(2-aminoethanethiol hydrochloride) through the allyl group to form aterminal amino group R, such as by free radical conditions such as theexposure to UV or other irradiation and/or by addition of peroxides, azocompounds, etc., e.g., as illustrated in the review of Griesbaum, K.,Angew. Chem. Internat. Edit, Vol.9, No.4, 273-287 (1970). Thereafter,amino group R is converted to a function capable of binding to asubstrate under free radical conditions e.g., a vinyl group. Asillustrated in the reaction scheme (5), the first step of thisconversion is the interaction of amino cryptand 2 with4-vinylbenzaldehyde. A Schiff base is the intermediate product of thisreaction (not shown on the scheme). On the second step, Schiff base isreacted in situ with NaBH₄ to give [2.2.2] cryptand 3 functionalizedwith polymerizable styryl moiety. This approach allows ready conversionof macrocycles into functionalized molecules for their further covalentattachment or incorporation into or onto various substrates using freeradical process such as grafting and coating.

In the foregoing description, C is functionalized by free radicaladdition of thiols (schemes (4) and(5)). Advantages of this processinclude the following:

-   1. Synthesis of allyl cryptand 1 described in prior art is the    superior method to build the macrocyclic framework of [2.2.2] 1    cryptand having pendant function for further attachment. The use of    allyl group alleviates the need for protection/deprotection steps    during the synthesis of the cryptands. Most of the prior art    examples of cryptand functionalization are based on protected    intermediates with longer routes for their synthesis effecting the    total outcome of the process. The method developed on a base of the    allyl precursor allows 100-200 g scale production of the    functionalized [2.2.2] cryptand. This is unusually large amount for    all described methods of the cryptand synthesis.-   2. The methods for a conversion of allyl cryptand to more reactive    functional molecules are limited. For instance, the authors who    first synthesized [2.2.2] allylic cryptand failed to convert the    allyl group to hydroxy group. The thiol addition was found very    effective for chemical transformation of relatively inert allyl    group to reactive amine. The amino group itself is highly efficient    for the functionalization of the substrates, however the    requirements for the anion exchange stationary phases are in favor    of the materials functionalized under conditions of radical    polymerization.-   3. Allylic group has lower reaction ability compared to styrenic    fragment under conditions of radical polymerization. Thus, allylic    monomers very often do not provide the required grafting efficiency    and lead to low capacity stationary phases. The developed thiol    addition allowed efficient transformation of the allyl group to    styrenic moiety via two step process (5). Allylic cryptand 1    converted to a styrenic derivative 3 now can be efficiently grafted    from the surface of the support providing novel high capacity anion    exchange stationary phase.-   4. Chemical stability of the stationary phases used in ion exchange    chromatography is of great importance. The extreme pHs at which the    ion exchange chromatography is performed impose considerable    limitations on chemistry of the functional monomers and linkers    connecting the ion exchange sites with the stationary phase. The    developed thiol addition method followed by grafting polymerization    provides extremely stable anion exchange materials on a base of the    cryptand functionalized resins. These stationary phases can be    operated at pH 1-14 at elevated temperatures. The ruggedness and    reproducibility of these phases after subjecting them to such harsh    conditions are superior to similar characteristics of the existing    anion exchange materials.

In the reaction scheme (5), the illustrated R group is NH₂ which reactsto form styrenic [2.2.2] cryptand 3 in which the pendant vinyl group canform a covalent bond with a corresponding vinyl group on a copolymerresin support substrate under free radical conditions as illustrated inExample 3 and FIG. 1.

One advantage of the foregoing reaction schemes is that individualmultiple (e.g., about 4×10¹⁴ to 4×10¹⁶ or more) strands of derivatizedmacrocycle compounds (e.g., cryptands) per square meter can be bound toeach support particle and projecting therefrom as illustrated in FIG. 1.This provides a substantial quantity of macrocycle compounds in a formatwhich is readily accessible to the approach of both cationic and anionicspecies in an aqueous environment. Prior art based on adsorbedmacrocycle compounds provides very limited capacity due to therestriction that this species must be adsorbed as a monolayer on ahydrophobic surface. Presenting the macrocycle compounds as a brushpolymer extending into the aqueous solution surrounding each supportparticle, permits significantly better diffusion kinetics than can beobserved with adsorbed monolayer coatings of macrocycle compounds whileat the same time allowing for significantly higher capacity than ispossible with an adsorbed monolayer coating. Furthermore, positioningthe macrocycle compounds in the proximity of a hydrophobic surfacecompromises the chromatographic performance of materials based onadsorbed macrocycle compounds. Anionic hydrophobic compounds exhibitpoor chromatographic efficiency when that retention site is located inthe proximity of a hydrophobic surface unless organic solvent is addedto the mobile phase. Of course, incorporating an organic solvent intomobile phase flowing past the support particles is incompatible with theprior art adsorbed monolayer coatings because such organic solvent willslowly wash the adsorbed coating away. While the composite materialderived from support particles which had been covalently derivatizedwith macrocycle brush polymers are compatible with organic solvents, thehigh cost of organic solvents and the high cost of disposal of organicsolvents make the use of organic solvents highly undesirable. Thus, thebrush polymer configuration described above provides superiorchromatographic properties without the need for the addition of organicsolvent and the incumbent disadvantages of its use.

Adsorbed macrocycle monolayer coatings of the prior art have been shownto be useful with macrocycle ion binding constants as low as 60.However, surprisingly similar macrocycles when constructed in the formof a covalently attached brush polymer coating failed to exhibit anyuseful ion binding characteristics in 100% aqueous environments.Apparently, applying a macrocycle as an adsorbed monolayer coating on ahydrophobic surface exposes the macrocycle to a substantially lowerdielectric environment than the aqueous environment above the surface.As such, ion binding affinities of adsorbed monolayer coatings aresubstantially higher than the equivalent macrocycle in a 100% aqueousenvironment. Therefore, in order to produce a useful material in thehighly desirable covalently bound brush polymer format, macrocycles musthave higher ion binding constants in order to provide useful ion bindingcharacteristics in 100% aqueous environments.

The vinyl moiety of the styrenic terminal group is readily attachedunder free radical conditions to polymeric substrate particles includingterminal groups with ethylenic unsaturation as described in U.S. Pat.No. 5,865,994 to form structure (3).

In the specific two-step reaction (scheme 5) discussed above for bondingR to the macrocycle, R is illustrated in the form of an amine convertedto an unsaturated carbon-carbon bond, specifically a vinyl group. Otherterminal functional groups may be employed so long as they can becovalently bonded to a desired support substrate.

According to the invention, one useful effective composition is aderivatized macrocycle compound with sufficient ion exchange propertiesto separate charged molecules. A particularly effective derivatizedmacrocycle compound of this type is a cryptand bound to a supportsubstrate with anion exchange properties. By anion exchange propertiesis meant the capability of performing anion exchange chromatography.Cryptands are particularly effective for anion separation because theyprovide ion binding characteristics sufficient for binding alkaline andalkaline earth metals under alkaline conditions and yet readily releasethese ions under acidic conditions, providing a convenient means ofconverting the cryptand from one alkali metal form to another.Comprehensive Supramolecular Chemistry Vol. 1-10, Jean-MarieLehn—Chairman of the Editorial Board, 1996 Elsevier Science Ltd; Izatt,R. M., et al., Chemical Reviews 91:1721-2085 (1991)). Significantlyhigher affinity of the cryptands to alkaline metal ions as compared tothe same property of the crown ethers for instance, provides better ionretention characteristics to the anion exchange stationary phasesfunctionalized with the cryptand modifiers. In fact, due to lowcapacities of the crown ether based materials they are impractical foruse as the anion exchange chromatographic phases with 100% aqueouseluents. Most of the industrial applications for ion exchangechromatographic processes are developed with the aqueous eluents,therefore the cryptand functionalized materials are superior to otherknown metal ion complexing agents.

The anion exchange capacity of the above cryptand-grafted resin canrange from about 15 to 2000 microequivalents per gram with a preferablerange from about 100 to 300, more preferably from about 120 to 225 andyet more preferably from about 150 to 200 microequivalents per gram.

One suitable support particle is a macroporous polymeric resin, e g,vinylbenzene ethylene, cross-linked with divinylbenzene. Suitablemacroporous resins are illustrated in U.S. Pat. No. 4,224,415.

In another embodiment of the invention, a hydrophilic layer may beattached to Z which forms the covalent attachment with the derivatizedmacrocycle. This has the advantage of reducing hydrophobic interactionbetween hydrophobic analytes and the surface to which the macrocyclesare covalently attached. This reduces the need for addition of solventto the mobile phase which as noted above is undesirable. Suitableprocedures are set forth in Examples 3 and 5.

The foregoing description illustrates functionalizing the cryptand toinclude unsaturation for covalent binding to Z by a thiol reaction.However, such functionalizing can also be accomplished by other. Otherelements besides sulfur can be attached to unsaturated carbon-carbonbonds under proper conditions of radical initiation. These elements aresilicon and germanium atoms having at least one hydrogen atom bound tothem directly. Substituted carbons, for example, halogenated carbons canalso be added to unsaturated carbon-carbon bonds via radical process.Elements mentioned above can be a part of the molecular structure (1)where sulfur is substituted for one of these elements bound directly orindirectly to R.

All patents and publications referred to herein are incorporated byreference.

Further details of the invention are illustrated in the followingnon-limiting examples.

EXAMPLE 1

This example describes a two-stage synthesis of a derivatized cryptandaccording to reaction scheme (5).

Methods for Derivatization of Allyl Cryptand 1 Procedure for theSynthesis of Cryptands 2 and 3 Scheme (5)

The procedure for the synthesis of allylic [2.2.2] cryptand is based onthe reported method (Babb, D. A., et al., Journal of HeterocyclicChemistry 23:609-613 (1986)). 12 g of allyl-derivatized cryptand wasdissolved in 70 ml of ethanol and 12 g of 2-aminoethanethiolhydrochloride was added to the reaction mixture. Reactor was purged withnitrogen. Solution was brought to reflux and 65 mg of AIBN was added. UVirradiation at 254 nm wave length was applied. Reaction mixture wasstirred under reflux and irradiated for eight hours. After every twohour period, a new portion of AIBN (65 mg) was added to the reactionmixture. Reaction is being monitored using TLC on neutral aluminum oxideand CH₂Cl₂/THF/MeOH; 10/5/1 as eluent.

Solvent was evaporated under reduced pressure. The rest was dissolved in100 ml of water. Lithium hydroxide was added to the aqueous solution toreach pH 11. The resulted solution was extracted three times with 100 mlof dichloromethane. Organic layer was extracted with 20% aqueous lithiumhydroxide and water and dried over anhydrous sodium sulfate. Afterevaporation of the solvent, crude aminocryptand 2 was dissolved in 200ml of methanol. To resulted solution, 12 g of 4-vinylbenzaldehyde in 40ml of methanol was added over a 1 hour period. Reactants were refluxedin methanol for 6 hours in presence of 10 mg of 4-t-butylcatechol.Methanol solution was filtered and cooled down to −5° C. 10 g of sodiumborohydride was added slowly to resulted solution. The reaction wascontinued under reflux for 24 hours. Methanol was evaporated underreduced pressure and the residue was mixed with 80 ml of water; pH wasbrought to 1.5 with ice cold 30% methanesulfonic acid. The resultedsolution was extracted three times with 150 ml of ether. Aqueous layerwas brought to pH 11 with lithium hydroxide and extracted three timeswith 100 ml of dichloromethane. Combined organic fractions wereextracted with water, dried over sodium sulfate and filtered. Solventwas evaporated under reduced pressure.

EXAMPLE 2

This example describes functionalizing the support substrate Z bydepositing a hydrophilic layer for binding to a derivatized cryptand.

Hydrophilic Layer Formed on Surface of Polymeric Particles Suitable forGrafting of Cryptand Monomer

2.3 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 3.3 gof tertiary butyl alcohol (Fluka). To this slurry was added 0.37 g ofvinylbenzylacetate made in house, 1 g of vinylacetate (Aldrich) and0.092 g of Vazo 64 initiator (Dupont). The entire material was dispersedhomogeneously and then placed in an oven at 60° C. for 18 hours. Theresultant polymeric material was washed with water, acetone, water andfinally with acetone. After hydrolysis, this material is now ready forgrafting with a cryptand monomer as shown in Example 3 below.

EXAMPLE 3

This example describes binding of the derivatized cryptand of Example 1to the functionalized Z of Example 2.

Cryptand Monomer is Attached to Polymeric Particles Suitable for Use asa Packing

2.35 g of a dried 55% cross-linked macroporous resin with preformedhydrophilic layer (substrate is ethylvinylbenzene cross-linked with 55%divinylbenzene, resin preparation described in U.S. Pat. No. 4,224,415)was dispersed in 3.4 grams of water and 0.5 g of 0.1 M methanesulfonicacid was added. To this slurry was added 0.5 g of cryptand monomer and0.2 g of azobiscyanopentanoic acid (Fluka). The entire material wasdispersed homogeneously and then placed in an oven at 50° C. for 20hours. The resultant polymeric material from above was washed withacetone followed by methanol, water, and 1M potassium hydroxide. Theresin was then packed in an analytical column using standard methods andapparatus at 6000 psi for 15 minutes. This polymeric column is suitablefor chromatographic separations of anionic species.

EXAMPLE 4 Cryptand Monomer is Attached to Polymeric Particles Which HaveNo Hydrophilic Layer Pre-Attached

In this example, the derivatized cryptand of Example 1 is bound to themacroporous resin starting material of Example 2 (without forming thehydrophilic layer).

EXAMPLE 5 Alternate Hydrophilic Layer Formed on Surface of PolymericParticles Suitable for Grafting of Cryptand Monomer

2.3 g of a dried 55% cross-linked macroporous resin (substrate isethylvinylbenzene cross-linked with 55% divinylbenzene, resinpreparation described in U.S. Pat. No. 4,224,415) was dispersed in 3.3 gof tertiary butylalcohol (Fluka). To this slurry was added 2.4 g ofvinylbenzylacetate (made in house), 0.092 g of Vazo 64 initiator(Dupont). The entire material was dispersed homogeneously and thenplaced in an oven at 60° C. for 18 hours. The resultant polymericmaterial was washed with water, acetone, water and finally with acetone.After hydrolysis, this material is now ready for grafting with acryptand monomer as described in Example 3 above.

EXAMPLE 6

This example shows the selectivity changes that can be accomplished bychanging the eluent cation (mobile phase).

Capacity vs. Cation Form

The conditions for these chromatograms shown in FIG. 2 are: Eluent: 70millimolar for each case, FIG. A potassium, FIG. B sodium, FIG. Clithium; flow rate, 1.0 mL/min; injection volume, 25 μL; temperature, 35deg C.; Detector, Suppressed Conductivity, ASRS®-ULTRA 4-mm, ExternalWater mode with Anion trap column (4×35 mm); Peaks: 1. Fluoride, 2 mg/L;2. Chloride, 3 mg/L; 3. Sulfate, 5 mg/L; 4. Nitrite, 10 mg/L; 5.Bromide, 10 mg/L. The column was a cryptand column of the type describedin the examples with dimensions of 4 mm I.D.×250 mm length. The particlesize was 10 micron.

EXAMPLE 7

This example shows adjustment of capacity during an analysis by using ahigher capacity mobile phase at the beginning and then changing to alower capacity format by either a step change or a gradient. As shown inFIG. 3, the initial mobile phase is sodium hydroxide and then a stepchange is made to a lithium hydroxide mobile phase, which is a very lowcapacity form. The first peaks eluting off are low valencypolyphosphates and the run ends with the elution of high valency ions.The conditions for Example 7 are as follows:

Polyphosphates: Capacity Gradient Prototype 3 × 150, 5μ Eluent: 30 mMNaOH for 2 minutes, step to 30 mM LiOH Inj. Volume: 5 μL Temperature:35° C. Detection: Suppress Conductivity ASRS-ULTRA 2-mm External watermode with ATC Sample: Polyphosphoric Acid 0.1%

The benefit of this approach is that it allows one to elute very highvalency ions without having to increase the mobile phase ionicconcentration. The total ionic concentration throughout the run is only30 mM, a relatively low concentration. This technique is known as“gradient capacity ion chromatography” (Lamb, et al. (Editors),“Variable Capacity Columns for Gradient Elution Anion ChromatographyBased on Macrocyclic Complexes,” Advances in Ion Chromatography, Vol. 2,Century International, Franklin, Mass., pp 215-231 (1990); Lamb andSmith, “Review: Application of Macrocyclic Ligands to IonChromatography,” J. Chromatogr. 546:73-88 (1991)).

Traditional anion exchange columns require very high concentrationmobile phases when analyzing polyvalent ions such as polyphosphates,oligosacharrides, oligonucleotides, etc. The concentrations neededsometimes exceed the ability of a suppressor to suppress. By using acryptand-based column one can eliminate large baseline changes oftenseen with steep gradients or step changes with conventional approaches.

1. An ion exchange composition formed by reacting unsaturated carbon tocarbon moieties pendant from derivatized ion binding cryptands withsupport substrates under free radical activation conditions to formcovalent bonds between said support substrates and said cryptandsthrough said carbon to carbon moieties, multiple cryptands projectingfrom each of said support substrates as strands of a brush polymer, saidion exchange composition having an ion binding constant of at leastabout 1000 in water.
 2. The ion exchange composition of claim 1 in whichsaid covalent bonds are formed between said cryptand pendant moietiesand unsaturated carbon to carbon moieties pendant from said supportsubstrates.
 3. The ion exchange composition of claim 1 in which saidsupport substrates comprise an organic polymer.
 4. The ion exchangecomposition of claim 1 in which said cryptand pendant carbon to carbonmoieties are on styrenic groups.
 5. The ion exchange composition ofclaim 1 in which the only ion binding moieties bound to said supportsubstrates are cryptands.
 6. An ion exchange composition formed byreacting unsaturated carbon to carbon moieties pendant from derivatizedion binding cryptands with support substrates under free radicalactivation conditions to form covalent bonds between said supportsubstrates and said cryptands through said carbon to carbon moieties,multiple cryptands projecting from each of said support substrates asstrands of a brush polymer, said support substrates being in the form ofa particulate resin bed comprising a chromatographic separation packedbed.
 7. The ion exchange composition of claim 6 in which particles insaid packed bed include at least about 100 microequivalents per gram. 8.An ion exchange composition formed by reacting unsaturated carbon tocarbon moieties pendant from derivatized ion binding cryptands withsupport substrates under free radical activation conditions to formcovalent bonds between said support substrates and said cryptandsthrough said carbon to carbon moieties, multiple cryptands projectingfrom each of said support substrates as strands of a brush polymer, saidstrands being bound to said support substrates and projecting therefromat a concentration of at least about 4×10¹⁴ cryptand strands per squaremeter of support substrates.